Human neural stem cells (NSCs), modified to express a therapeutic transgene, hold great promise for brain tumor therapy due to their inherent tumor-tropic properties and potential use as vehicles for delivering chemotherapy directly to infiltrating glioma cells in the brain. NSCs can overcome obstacles of drug delivery that limit current gene therapy strategies to provide an effective anti-tumor response. Data from animal models have demonstrated the safety and efficacy of NSCs for tracking to invasive tumor cells as well as to distant micro-tumor foci and delivering therapeutic gene products to tumor cells. Our first-in-human pilot study of cytosine deaminase (CD)-expressing NSCs given in combination with oral 5-fluorocytosine (5-FC) demonstrated that one dose of NSCs followed by a 7-day course of 5-FC in recurrent high-grade glioma patients was safe and feasible. With intracerebral micro dialysis, we documented proof-of-concept-that the NSCs converted the prodrug 5-FC to 5-fluorouracil in the brain. Results of immunologic correlative studies showed no evidence of NSC immunogenicity after first exposure. Autopsy data provided evidence that NSCs migrated to distant tumor sites and did not divide and form secondary tumors. We have now developed a second generation of NSCs that secrete a modified form of the enzyme carboxylesterase (hCE1m6), which more efficiently converts irinotecan to its more powerful metabolite SN-38. Based on strong preclinical efficacy and safety data, we plan to perform a phase I study of intracranially administered hCE1m6 -NSCs in combination with intravenous irinotecan. Using a standard 3 + 3 dose escalation schema, the primary objective of this study will be to define the phase II recommended doses of this neural stem cell-based treatment in patients with recurrent high grade gliomas (Specific Aim 1). Other important study objectives will be to use intracerebral micro dialysis to establish proof-of-concept regarding the ability of these hCE1m6-NSCs to produce high concentrations of SN-38 locally in the brain (Specific Aim 2), and perform immunologic correlative studies to assess for the possible development of immune responses to these NSCs in study participants (Specific Aim 3).